PEATmoss (Physcomitrella Expression Atlas Tool): a unified gene expression atlas for the model plant Physcomitrella patens

Physcomitrella patens is a bryophyte model plant that is often used to study plant evolution and development. Its resources are of great importance for comparative genomics and evo‐devo approaches. However, expression data from Physcomitrella patens were so far generated using different gene annotation versions and three different platforms: CombiMatrix and NimbleGen expression microarrays and RNA sequencing. The currently available P. patens expression data are distributed across three tools with different visualization methods to access the data. Here, we introduce an interactive expression atlas, Physcomitrella Expression Atlas Tool (PEATmoss), that unifies publicly available expression data for P. patens and provides multiple visualization methods to query the data in a single web‐based tool. Moreover, PEATmoss includes 35 expression experiments not previously available in any other expression atlas. To facilitate gene expression queries across different gene annotation versions, and to access P. patens annotations and related resources, a lookup database and web tool linked to PEATmoss was implemented. PEATmoss can be accessed at https://peatmoss.online.uni-marburg.de     

Physcomitrella patens Reute mCherry as a tool for efficient crossing within and between ecotypes

• Physcomitrella patens is a monoecious moss that is predominantly selfing in the wild. Laboratory crossing techniques have been established and crosses between the sequenced Gransden ecotype and the genetically divergent Villersexel ecotype were used for genetic mapping. The recently introduced ecotype Reute has a high fertility rate and is genetically more closely related to the Gransden ecotype than the Villersexel ecotype. Reute sexual reproduction phenology is similar to Gransden, which should allow successful crossing.
• Using the Reute ecotype and an existing Gransden mutant as a test case, we applied a normalised crossing approach to demonstrate crossing potential between these ecotypes. Also, using a standard transformation approach, we generated Reute fluorescent strains expressing mCherry that allow an easy detection of crossed offspring (sporophyte).
• We show that Reute can be successfully crossed with a self‐infertile DR5:DsRed2 mutant generated in the Gransden background. Using newly established Reute fluorescent strains, we show that they can efficiently fertilise Reute as well as Gransden wild type. The resulting progeny display Mendelian 1:1 segregation of the fluorescent marker(s), demonstrating the suitability of such strains for genetic crossing.
• Overall our results demonstrate that Reute is highly suitable for genetic crossing. The Reute mCherry strain can be used as a suitable background for offspring selection after crossing.

     

Immunohistochemical analysis of DNA 'mismatch-repair' enzyme human Mut-S-Homologon-2 in ovarian carcinomas

The human Mut-S-Homologon-2 (hMSH-2) gene product is a member of a highly conserved family of proteins involved in postreplication mismatch repair. We have analysed hMSH-2 expression in normal ovarian tissue (n=15) and ovarian carcinomas (n=40). hMSH-2 protein was investigated immunohistochemically on frozen sections using a highly sensitive streptavidin–peroxidase technique and a specific mouse monoclonal antibody (clone FE11). A hMSH-2-immunoreactivity score (hMSH-2-IRS) for semiquantitative analysis of hMSH-2 expression is presented. In normal ovarian tissue, we only found weak nuclear immunoreactivity for hMSH-2 in 60%, while the remaining 40% were hMSH-2 negative (mean hMSH-2-IRS: 0.73; SD: ±0.70). All ovarian carcinomas analysed revealed moderate to strong nuclear immunoreactivity (mean hMSH-2-IRS: 8.05; SD: ±3.65). hMSH-2 staining was heterogeneous, with visual differences between individual tumour cells. Expression of hMSH-2 protein was consistently and strongly upregulated in tumour cells of ovarian carcinomas as compared to normal ovarian tissue. No statistically significant correlation in comparing the labelling patterns for hMSH-2 with the labelling patterns for Ki-67 (mean percentage of Ki-67 positive tumour cells: 25.88%; SD: ±18.43) was observed in ovarian carcinomas. Furthermore, no statistical significant correlations between hMSH-2-IRS and histological grading (p=0.47), histological type of carcinoma (p=0.706) or FIGO-classification (p=0.054) were found. Our findings indicate that (a) hMSH-2 is expressed in normal human ovarian tissue, (b) expression of hMSH-2 is increased in ovarian carcinomas, (c) expression of hMSH-2 may be of importance for the genetic stability of ovarian carcinomas in vivo, (d) hMSH-2 mutations may not cause microsatellite instability in ovarian carcinomas, (e) hMSH-2 may contribute to mechanisms responsible for resistance to anticancer drugs.     

Immunohistochemical Analysis of DNA ‘mismatch-repair’ Enzyme Human Mut-S-Homologon-2 in Ovarian Carcinomas

The human Mut-S-Homologon-2 (hMSH-2) gene product is a member of a highly conserved family of proteins involved in postreplication mismatch repair. We have analysed hMSH-2 expression in normal ovarian tissue (n=15) and ovarian carcinomas (n=40). hMSH-2 protein was investigated immunohistochemically on frozen sections using a highly sensitive streptavidin–peroxidase technique and a specific mouse monoclonal antibody (clone FE11). A hMSH-2-immunoreactivity score (hMSH-2-IRS) for semiquantitative analysis of hMSH-2 expression is presented. In normal ovarian tissue, we only found weak nuclear immunoreactivity for hMSH-2 in 60%, while the remaining 40% were hMSH-2 negative (mean hMSH-2-IRS: 0.73; SD: ±0.70). All ovarian carcinomas analysed revealed moderate to strong nuclear immunoreactivity (mean hMSH-2-IRS: 8.05; SD: ±3.65). hMSH-2 staining was heterogeneous, with visual differences between individual tumour cells. Expression of hMSH-2 protein was consistently and strongly upregulated in tumour cells of ovarian carcinomas as compared to normal ovarian tissue. No statistically significant correlation in comparing the labelling patterns for hMSH-2 with the labelling patterns for Ki-67 (mean percentage of Ki-67 positive tumour cells: 25.88%; SD: ±18.43) was observed in ovarian carcinomas. Furthermore, no statistical significant correlations between hMSH-2-IRS and histological grading (p=0.47), histological type of carcinoma (p=0.706) or FIGO-classification (p=0.054) were found. Our findings indicate that (a) hMSH-2 is expressed in normal human ovarian tissue, (b) expression of hMSH-2 is increased in ovarian carcinomas, (c) expression of hMSH-2 may be of importance for the genetic stability of ovarian carcinomas in vivo, (d) hMSH-2 mutations may not cause microsatellite instability in ovarian carcinomas, (e) hMSH-2 may contribute to mechanisms responsible for resistance to anticancer drugs.     

LAD-Ligation: A Murine Model of Myocardial Infarction

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.Download video file.^{(98M, mp4)}     

Selective staining of fungal hyphae in parasitic and symbiotic plant-fungus associations

A cytochemical method for light microscopical studies is described which allows the specific detection of fungal hyphae in plant-fungus associations: e.g. lichens, mycorrhiza, or fungal infections of plant tissue. The specimens were fixed and embedded in epoxy resin by a standard protocol for electron microscopy. Semithin sections were successively incubated with fluorescein isothiocyanate labelled wheat germ agglutinin (FITC-WGA) and calcofluor white (CW). FITC-WGA stained exclusively the fungal cell walls while CW stained both the fungal and the plant cell walls. Therefore, FITC-WGA is an excellent marker for the fungal hyphae.